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Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork.

Junk AK, Goel M, Mundorf T, Rockwood EJ, Bhattacharya SK - Mol. Vis. (2010)

Bottom Line: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively.HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein.Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA. ajunk@med.miami.edu

ABSTRACT

Purpose: To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls.

Methods: Tissue specimens were obtained from trabeculectomy (n=45 open angle glaucoma; Caucasian, average age 61+/-8 years of age of both genders) and from cadaver eyes (n=15 control and n=5 glaucoma; Caucasian, average age 63+/-4 years of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of L-malate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm.

Results: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS-PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls.

Conclusions: Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

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Western analysis for lipid peroxidation product (4-hydroxynonenal; HNE) modification of isocitrate dehydrogenase. Trabecular meshwork protein extract (5 µg) from each eye with donor age and gender as indicated (M, male; F, female) was loaded on each lane and separated over a 4%–20% SDS–PAGE. The proteins were transferred onto a PVDF membrane and probed using polyclonal antibodies to isocitrate dehydrogenase (isocitrate DH). The corresponding protein bands from the region that showed immunoreactive for isocitrate dehydrgenase was excised from an identical gel and electrophoresed and subsequently transferred on a PVDF membrane and probed for isocitrate dehydrogenase (isocitrate DH) against 4-hydroxynonenal (HNE) and against 3-glyceralde dehydrgenase (GAPDH) as indicated.
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f2: Western analysis for lipid peroxidation product (4-hydroxynonenal; HNE) modification of isocitrate dehydrogenase. Trabecular meshwork protein extract (5 µg) from each eye with donor age and gender as indicated (M, male; F, female) was loaded on each lane and separated over a 4%–20% SDS–PAGE. The proteins were transferred onto a PVDF membrane and probed using polyclonal antibodies to isocitrate dehydrogenase (isocitrate DH). The corresponding protein bands from the region that showed immunoreactive for isocitrate dehydrgenase was excised from an identical gel and electrophoresed and subsequently transferred on a PVDF membrane and probed for isocitrate dehydrogenase (isocitrate DH) against 4-hydroxynonenal (HNE) and against 3-glyceralde dehydrgenase (GAPDH) as indicated.

Mentions: Lipid oxidation products have been implicated to cause damage to the aqueous drainage system [11]. Our SDS–PAGE analysis suggests the presence of HNE modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls (Figure 2). Two identical gel electrophoretic separations were performed, protein bands immunoreactive for isocitrate dehydrogenase were excised and subsequently submitted to electrophoresis in a second 4%–20% SDS–PAGE and probed with antibodies to isocitrate dehydrogenase and HNE. The identical gel was probed with antibodies to isocitrate dehydrogenase, HNE and glyceraldehyde-3-dehydrogenase (GAPDH). Western analysis also revealed presence of isocitrate dehydrogenase and other enzymes at relatively high molecular weights indicating aggregate formation as well as locations in gel corresponding to molecular weights lower than the bona fide molecular weights of the proteins consistent with degradation of the proteins (data not shown). However, these analyses necessitated much higher protein loads and longer exposure of detection reagents. While they were thus detected severe darkening of the film overall suggests relative low abundance of aggregated and degraded proteins. Such aggregated and degraded proteins were more frequent and abundant in glaucomatous than normal TM. It is important to note that modification of calpain-1 by isolevuglandin, another lipid peroxidation product was previously observed in the trabecular meshwork, and was associated with both aggregated and degraded calpain-1 [12].


Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork.

Junk AK, Goel M, Mundorf T, Rockwood EJ, Bhattacharya SK - Mol. Vis. (2010)

Western analysis for lipid peroxidation product (4-hydroxynonenal; HNE) modification of isocitrate dehydrogenase. Trabecular meshwork protein extract (5 µg) from each eye with donor age and gender as indicated (M, male; F, female) was loaded on each lane and separated over a 4%–20% SDS–PAGE. The proteins were transferred onto a PVDF membrane and probed using polyclonal antibodies to isocitrate dehydrogenase (isocitrate DH). The corresponding protein bands from the region that showed immunoreactive for isocitrate dehydrgenase was excised from an identical gel and electrophoresed and subsequently transferred on a PVDF membrane and probed for isocitrate dehydrogenase (isocitrate DH) against 4-hydroxynonenal (HNE) and against 3-glyceralde dehydrgenase (GAPDH) as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2904041&req=5

f2: Western analysis for lipid peroxidation product (4-hydroxynonenal; HNE) modification of isocitrate dehydrogenase. Trabecular meshwork protein extract (5 µg) from each eye with donor age and gender as indicated (M, male; F, female) was loaded on each lane and separated over a 4%–20% SDS–PAGE. The proteins were transferred onto a PVDF membrane and probed using polyclonal antibodies to isocitrate dehydrogenase (isocitrate DH). The corresponding protein bands from the region that showed immunoreactive for isocitrate dehydrgenase was excised from an identical gel and electrophoresed and subsequently transferred on a PVDF membrane and probed for isocitrate dehydrogenase (isocitrate DH) against 4-hydroxynonenal (HNE) and against 3-glyceralde dehydrgenase (GAPDH) as indicated.
Mentions: Lipid oxidation products have been implicated to cause damage to the aqueous drainage system [11]. Our SDS–PAGE analysis suggests the presence of HNE modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls (Figure 2). Two identical gel electrophoretic separations were performed, protein bands immunoreactive for isocitrate dehydrogenase were excised and subsequently submitted to electrophoresis in a second 4%–20% SDS–PAGE and probed with antibodies to isocitrate dehydrogenase and HNE. The identical gel was probed with antibodies to isocitrate dehydrogenase, HNE and glyceraldehyde-3-dehydrogenase (GAPDH). Western analysis also revealed presence of isocitrate dehydrogenase and other enzymes at relatively high molecular weights indicating aggregate formation as well as locations in gel corresponding to molecular weights lower than the bona fide molecular weights of the proteins consistent with degradation of the proteins (data not shown). However, these analyses necessitated much higher protein loads and longer exposure of detection reagents. While they were thus detected severe darkening of the film overall suggests relative low abundance of aggregated and degraded proteins. Such aggregated and degraded proteins were more frequent and abundant in glaucomatous than normal TM. It is important to note that modification of calpain-1 by isolevuglandin, another lipid peroxidation product was previously observed in the trabecular meshwork, and was associated with both aggregated and degraded calpain-1 [12].

Bottom Line: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively.HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein.Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA. ajunk@med.miami.edu

ABSTRACT

Purpose: To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls.

Methods: Tissue specimens were obtained from trabeculectomy (n=45 open angle glaucoma; Caucasian, average age 61+/-8 years of age of both genders) and from cadaver eyes (n=15 control and n=5 glaucoma; Caucasian, average age 63+/-4 years of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of L-malate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm.

Results: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS-PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls.

Conclusions: Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

Show MeSH
Related in: MedlinePlus